Phase Separation Behavior Modifying Agents for Aqueous Two-Phase Separation Within Porous Material

ABSTRACT

The present invention relates to a method and/or device for improving the separation behaviors and performance of aqueous two-phase system (ATPS) for the isolation and/or concentration of one or more target analytes from a sample. In one embodiment, the present method and device comprise ATPS components within a porous material and one or more phase separation behavior modifying agents that improve the separation behavior and performance characteristics of ATPS, including but not limited to the increasing the stability or reducing fluctuations of ATPS thought the adjustment of total volume of a sample solution that undergoes phase separation, volume ratio of the two phases of the ATPS, fluid flow rates, and concentrations of ATPS components.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 16/608,842filed on Oct. 27, 2019, which is the national stage of InternationalApplication No. PCT/US2018/35569 filed on Jun. 1, 2018, which claims thebenefit of U.S. Provisional Application No. 62/513,994, filed Jun. 1,2017 and U.S. Provisional Application No. 62/599,001, filed Dec. 14,2017. The entire contents and disclosures of the preceding applicationare incorporated by reference into this application.

Throughout this application, various publications are cited. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

FIELD OF THE INVENTION

The present invention relates to a method and/or device for improvingthe behaviors and performance of aqueous two-phase system (ATPS) withina porous material for isolating and/or concentrating one or more targetanalytes from a sample solution. The present method and device arerelated to using one or more phase separation behavior modifying agentsto improve the separation behavior and performance characteristics ofATPS components within a porous material. A modifying agent can increasethe stability or reduce fluctuations of ATPS by affecting the volumesand volume ratio of the two phases after separation, fluid flow rates,and/or concentrations of the ATPS components. In one embodiment, therange of concentrations of the ATPS components for phase separation iswidened significantly when a modifying agent(s) is added to the ATPS,thereby increasing the yield of a purified target analyte. The presentinvention further provides a device for carrying out the method of thepresent invention.

BACKGROUND OF THE INVENTION

The concentration of a target analyte is a critical parameter in manyresearch and diagnostic applications. A purified target analyte must beof high quality and sufficient quantity, such that it can be used invarious downstream applications including detection, clinical diagnosisand so on. Obtaining a target analyte in a purified form is acomplicated task due to the presence of large amounts of cellularmaterials and macromolecules (e.g. proteins and carbohydrates) (alsotermed as ‘polymers’) in such samples as urine, blood, plasma, serum,saliva and other biological fluids.

For target analytes that are present at very low concentrations inbiological fluids, such as urine and blood, there is a need to obtainlarge volumes of biological fluids in order to obtain sufficientquantity of the target analytes for subsequent detection by moleculartechniques.

It has been a huge challenge to detect the existence of an analyte whichhas an extremely low concentration. The analyte can be a biomarker of adisease such as a cell free DNA (cfDNA), circulating tumor DNA (ctDNA)or a protein which exists in a sample such as saliva, blood, urine andother bodily fluids of a patient. Many of the existing diagnostic ordetection methods may falsely report that the analyte does not exist ifthe analyte concentration is too low. For instance, the gold standard ofdiagnostics such as Polymerase Chain Reaction (PCR) and Enzyme-LinkedImmune Sorbent Assay (ELISA) may produce a false negative result if thetarget analyte has extremely low quantity beyond the detection limit ofthe assay.

Despite discovery and invention of new biomarkers for many diseases inrecent years, diagnostic tests based on these new biomarkers, such ascfDNA testing, have not been adopted in routine clinical procedures dueto lack of sensitivity and specificity. Where biomarkers are present inlow quantities, accurate detection hinges upon isolation methods thatcan concentrate the biomarkers from background. Depending on theisolation method, this challenge can be complicated with variance infragment size and influences by test inhibitors.

There are methods described in the literature to concentrate targetanalytes. A method to concentrate an analyte by porous materialdehydrated with ATPS has been disclosed in patent publicationWO2017041030. However, the number of folds of concentration is rathersmall to satisfy the need in practice. Despite the popularity of ATPS,it is still not widely used in the industry due to its limitations. Forexample, it is limited by the maximum sample volume the system canprocess, the minimum and maximum concentration of ATPS components, andthe yield of purified target analytes. Specifically, the performance ofATPS is not very stable during phase separation due to fluctuations insample volumes, concentration and ratio of ATPS components, and so on,thereby affecting the purification of the target analytes in terms ofefficiency, purity and yield.

To overcome these limitations, the present invention provides animproved method and device to concentrate and purify target analytesfrom samples for further analysis. Using ATPS components which areembedded within a porous material and one or more phase separationbehavior modifying agents, the present methods and devices canconcurrently perform a number of tasks including isolating targetanalytes, removing non-target analytes and impurities, and concentratingtarget analytes in high yield in any suitable condition, so that themethods and devices can be implemented using a wide range ofconcentrations of ATPS components, resulting in stable phase separationduring the whole process with no or minimal fluctuations, and underdifferent flow rates. In short, the present invention significantlyimproves the behaviors and performance of ATPS. The present inventionfurther provides a device for carrying out the method of the presentinvention.

SUMMARY OF THE INVENTION

The foregoing background, as well as the following detailed description,are better understood when read in conjunction with the appendedfigures. The figures are intended to be illustrative and not limiting.The disclosure is not limited to the precise arrangements and examplesshown herein.

The present invention relates to a method and/or device for improvingthe behaviors and performance of an aqueous two-phase system (ATPS)within a porous material for isolating or concentrating a target analytefrom a sample solution. In one embodiment, the present method and/ordevice comprise ATPS components within a porous material, and one ormore phase separation behavior modifying agents that improve theseparation behavior and performance characteristics of ATPS, includingbut not limited to increasing the stability or reducing fluctuations ofATPS through adjustment of parameters including but not limited to thetotal volume of a sample solution that undergoes phase separation,volume ratio, fluid flow rates and the concentrations of ATPScomponents.

In one embodiment, the present method and device comprise a porousmaterial embedded with ATPS components and one or more phase separationbehavior modifying agents that widen the range of concentrations of theATPS components effective for phase separation.

In one embodiment, the present method and device comprise a porousmaterial embedded with ATPS components and one or more phase separationbehavior modifying agents that increase the yield of purified targetanalytes.

In one embodiment, the present method and device comprise a porousmaterial embedded with ATPS components and one or more phase separationbehavior modifying agents that improve the stability of ATPS duringphase separation by minimizing fluctuations in the volumes and/or volumeratios of the two phases.

In one embodiment, the method and device comprise a porous materialembedded with ATPS components and one or more phase separation behaviormodifying agents to remove contaminants from a sample solution.

In one embodiment, the present invention provides a method and/or devicefor concentration of a target analyte in a solution using ATPScomponents which are embedded in a porous material, and one or morephase separation behavior modifying agents.

In one embodiment, phase separation behavior modifying agents arecompounds which can induce or promote phase separation by changing thetemperature, pH, salt concentration, hydrophobicity/hydrophilicity,surface tension, and/or ionic strength of the ATPS. As a result, thetarget analyte partitions preferentially into one of the two phases,depending on their characteristics. In one embodiment, phase separationbehavior modifying agents include compounds carrying an acidicfunctional group, an amine functional group, and/or both a hydrophilicgroup and a hydrophobic group.

In one embodiment, the present invention provides a device for isolatingor concentrating one or more target analytes from a sample solution, thedevice comprising:

-   -   i) a porous material;    -   i_i) components capable of forming a first phase solution and a        second phase solution of an aqueous two-phase system (ATPS); and    -   iii) one or more phase separation behavior modifying agents;

wherein said components and said phase separation behavior modifyingagents are embedded in said porous material, wherein when a solutioncontaining said target analytes flows through said porous material, anaqueous two-phase system comprising said first phase solution and saidsecond phase solution is generated, wherein the separation behavior ofthe two phase solutions is altered by said phase separation behaviormodifying agents, and said target analytes are isolated or concentratedon said porous material.

In one embodiment, the present invention provides a device for isolatingor concentrating one or more target analytes, the device comprising:

-   -   i) a porous material;    -   ii) components capable of forming a first phase solution and a        second phase solution of an aqueous two-phase system (ATPS); and    -   iii) one or more phase separation behavior modifying agents;

wherein one or more of said components and said phase separationbehavior modifying agents are embedded in said porous material, whereinthe balance of said components and said phase separation behaviormodifying agents are to be mixed with a solution containing said targetanalytes, thereby forming a mixed solution, wherein when said mixedsolution flows through said porous material, an aqueous two-phase systemcomprising said first phase solution and said second phase solution isgenerated, wherein the separation behavior of the two phase solutions isaltered by said phase separation behavior modifying agents, and saidtarget analytes are isolated or concentrated on said porous material.

In one embodiment, the present invention provides a method for isolatingor concentrating one or more target analytes from a sample solution, themethod comprising:

-   -   i) obtaining a sample solution containing the target analytes;        and    -   ii) contacting the sample solution with a porous material        embedded with one or more phase separation behavior modifying        agents, components capable of forming a first phase solution and        a second phase solution of an aqueous two-phase system (ATPS),        wherein, when a solution containing said target analytes flows        through the porous material, an aqueous two-phase system        comprising said first phase solution and said second phase        solution is generated,

wherein the separation behavior of the two phase solutions is altered bysaid modifying agents;

wherein the target analytes are isolated or concentrated on said porousmaterial.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 provides a few embodiments showing how a phase separationbehavior modifying agent can distribute within a rectangular porousmaterial. The phase separation behavior modifying agent may be disbursedwithin the porous material at many different locations and at differentconcentrations within each location.

FIG. 2 shows phase separation diagrams (binodal curves) under variousflow rates comparing the separation behavior and characteristics of ATPScomponents with or without behavior modifying agents, illustratingbenefits of phase separation behavior modifying agents on phaseseparation characteristics of the ATPS components. In some instancesthese phase separation diagrams represent a time progression of fluidflowing through a porous material embedded with ATPS components andthereby resolubilizing ATPS components that are dehydrated in thatmaterial. As time goes on, more ATPS components will get into solution.The curved lines are binodal curves which indicate the relativeconcentrations of ATPS components that are effective for phaseseparation, thereby setting a boundary between a monophasic system and adiphasic system. Under the theory of binodal curve, phase separationoccurs only when the relative concentration of the ATPS components hasreached a certain value. Any point outside the binodal curve representsa monophasic system (no phase separation), while any point inside thebinodal curve represents a diphasic system (phase separation). Thehorizontal solid line refers to a tie line which denotes conditionsunder which the two phases exist in equilibrium with each other at aparticular temperature. All systems on the tie line produce the same twophases at a given temperature and the intersections of the tie line withthe binodal curve represent the compositions of the two phases thatexist in equilibrium with each other at that temperature. The volumeratio of the ATPS solution can be determined by comparing the length ofthe point on the tie line to the binodal curve on the left to the lengthof the point to the binodal curve on the right (referred to as the“lever arm rule”). For example in Panel A of FIG. 2 , the volume ratioof the two phase solutions is 1:5 when the relative concentration ofATPS components is n, while in Panel B of FIG. 2 the volume ratio is 5:1when the relative concentration of ATPS components is 2n. In contrast,when the relative concentration of ATPS components is 3n falling outsidethe tie line, the two phase solutions will not undergo a phaseseparation and remain as one single phase (Panel C of FIG. 2 ).Accordingly, the partition coefficient of a target analyte is the samealong the tie line regardless of changes in the relative concentrationof ATPS components. Since the yield of the target analyte by ATPSdepends on the partition coefficient of the target analyte and thevolume of the target phase recovered, the yield will depend on the tieline and the position of the ATPS on that tie line. Typically, thelonger the tie line is, the higher the yield will be. The phase diagramson the right panel (Panels D-F of FIG. 2 ) indicated that addition ofphase separation behavior modifying agent to the ATPS shifts theposition of the binodal curve downward and thus increases the length ofthe tie line at any given temperature, thereby allowing phase separationto occur within a wider range of relative concentration of the two phasecomponents, as well as increasing the partition coefficient. As aresult, the range of ATPS concentration capable of phase separation iswidened significantly. Furthermore, in certain embodiments where theATPS components and the phase separation behavior modifying agents aredehydrated within the porous material, the phase separation volume ratiobecomes more stable with respect to flow rate and fluctuations in theconcentration of the ATPS components. Along the same tie line, anincrease in ATPS component concentration will result in more extremevolume ratios. Under the theory of binodal curve, if the operatingrelative concentration of ATPS components is too close to either side ofthe binodal curve, even a slight change of volume ratio of the two phasesolutions (e.g. a slight dilution or concentration of the solutions) cancause a shift of the operating relative concentration to another side ofthe binodal curve where only one phase exists. However, in cases wherethe porous material is embedded with both the ATPS components andmodifying agents in dehydrated form, the increase in ATPS componentconcentration also correlates with an increase in phase separationbehavior modifying agents, and therefore leading to a proportionaldownward shift of the binodal curve and a longer tie line at temperatureT as compared to the phase diagrams in Panels A-C of FIG. 2 . As such,the same relative concentration of ATPS components would have a smallerinfluence on the volume ratio when modifying agents are present, makingthe system more stable and robust. It should be noted that FIG. 2 isillustrative of a temperature dependent ATPS with a single ATPScomponent, however the concept described above similarly applies toATPSs that are not temperature dependent and ATPSs that have multipleATPS components.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, several embodiments of the invention aredescribed. For purposes of explanation, specific configurations anddetails are set forth in order to provide a thorough understanding ofthe embodiments. In addition, to the plural or singular forms of a wordand to the extent that orientations of the embodiments are described as,“top”, “bottom”, “front”, “back”, “left”, “right” and the like, thesewordings are to aid the reader in understanding the embodiments and arenot meant to be limiting physically. It is apparent to a person skilledin the art that the present invention may be practiced without specificdetails. The invention will be better understood by reference to theexamples which follow, but those skilled in the art will readilyappreciate that the specific examples are for illustrative purposes onlyand should not limit the scope of the invention which is defined by theclaims which follow thereafter. It is to be noted that the transitionalterm “comprising” or “including”, which is synonymous with “containing”or “characterized by”, is inclusive or open-ended and does not excludeadditional, unrecited elements or method steps.

The present invention relates to a method and/or device for improvingthe behaviors and performance of aqueous two-phase system (ATPS) withina porous material to isolate or concentrate a target analyte from asample solution. The present invention further provides a device forcarrying out the method of the present invention.

In one embodiment, among the many different possibilities contemplated,the present methods and/or devices take the advantage of the phaseseparation behavior modifying agents to improve one or more phaseseparation behaviors and performance characteristics of the ATPScomponents. In one embodiment, it is further contemplated thatimprovement in phase separation behaviors and performancecharacteristics of the ATPS components can improve the functionality andintegration of many other devices, methods, or practices being used inconjunction with the present porous material or device.

In one embodiment, the present invention provides a method and a devicefor isolating or concentrating a target analyte in a solution using ATPScomponents which are embedded on a porous material, and one or morephase separation behavior modifying agents. In one embodiment, phaseseparation behavior modifying agents are compounds which can induce orpromote phase separation by changing the temperature, pH, saltconcentration, and/or ionic strength of the ATPS system. As a result,the target analyte partitions preferentially into one phase or theother, depending on their characteristics.

Accordingly, the present invention provides an aqueous two-phase system(ATPS) which is capable of isolating or concentrating a target analytefrom a sample under a wide range of concentration of ATPS components.The present invention overcomes the limitations of the existingtechnologies such as limitations on sample size, minimum and maximumconcentrations of ATPS components and yield of purified target analytes.The present invention facilitates a faster and efficient distribution oftarget analytes in the aqueous two-phase system without the need forcomplex instrumentation and allows purification of target analytes fromlarge volume and complex biological materials with high yield, whileavoiding target analytes contamination.

In one embodiment, the present invention provides a device for isolatingor concentrating one or more target analytes from a sample solution, thedevice comprising:

-   -   i) a porous material;    -   ii) components capable of forming a first phase solution and a        second phase solution of an aqueous two-phase system (ATPS); and    -   iii) one or more phase separation behavior modifying agents;

wherein said components and said phase separation behavior modifyingagents are embedded in said porous material, wherein when a solutioncontaining said target analytes flows through said porous material, anaqueous two-phase system comprising said first phase solution and saidsecond phase solution is generated, wherein the separation behavior ofthe two phase solutions is altered by said phase separation behaviormodifying agents, and said target analytes are isolated or concentratedon said porous material.

In one embodiment, the present invention provides a device for isolatingor concentrating one or more target analytes, the device comprising:

-   -   i) a porous material;    -   ii) components capable of forming a first phase solution and a        second phase solution of an aqueous two-phase system (ATPS); and    -   iii) one or more phase separation behavior modifying agents;        wherein one or more of said components and said phase separation        behavior modifying agents are embedded in said porous material,        wherein the balance of said components and said phase separation        behavior modifying agents are to be mixed with a solution        containing said target analytes, thereby forming a mixed        solution, wherein when said mixed solution flows through said        porous material, an aqueous two-phase system comprising said        first phase solution and said second phase solution is        generated, wherein the separation behavior of the two phase        solutions is altered by said phase separation behavior modifying        agents, and said target analytes are isolated or concentrated on        said porous material.

In one embodiment, the present invention provides a method for isolatingor concentrating one or more target analytes from a sample solution, themethod comprising:

-   -   i) obtaining a sample solution containing the target analytes;        and    -   ii) contacting the sample solution with a porous material        embedded with one or more phase separation behavior modifying        agents, components capable of forming a first phase solution and        a second phase solution of an aqueous two-phase system (ATPS),        wherein, when a solution containing said target analytes flows        through the porous material, an aqueous two-phase system        comprising said first phase solution and said second phase        solution is generated, wherein the separation behavior of the        two phase solutions is altered by said phase separation behavior        modifying agents;

wherein the target analytes are isolated or concentrated on said porousmaterial.

In one embodiment, when ATPS is applied on a porous material (e.g.paper), the two phases of the ATPS separate from each other as the mixedphase solution flows through the porous materials. The resulting phasesolutions are of different physicochemical properties and each phasetravels through the porous matrix at different rates. Differentmolecules in a mixture would be distributed differentially between thetwo phase solutions due to their different properties, and it ispossible to separate and concentrate target molecules using ATPS withminimal set up and human intervention.

The advantage of the invention is that high concentration and high yieldof the target analyte can be obtained in a simple way and compatiblewith downstream application analysis without further step ofpurification or concentration.

The methods and devices provided herein are robust, inexpensive, simple,easy to handle, safe, user friendly and fast. The present method anddevice are able to purify and concentrate the target analyte and therebyensure the performance of the downstream applications using the purifiedand concentrated analyte will not be affected by impurities in theoriginal sample.

Furthermore, the present invention is applicable to samples containingthe target analyte in a very low amount, or of a small volume and isreadily adaptable to automation including high throughput screeningsystems.

In one embodiment, the present invention provides a method and/or devicecomprising ATPS as a concentration module linked with a lateral flowimmunoassay (LFA) component as a detection module. In one embodiment,the detection module is housed in a plastic housing with a viewingwindow and gold nanoprobes. As the sample wicks up the device and ATPScomponents undergo phase separation, the analytes are concentratedsubstantially in the leading front. Concentrated analytes are thendetected by the LFA module generating visual test results.

Type of Samples and Target Analytes

In one embodiment, the present method and device can separate a targetanalyte from non-target molecules (e.g., small molecules andmacromolecules which are typically of natural origin and may interferewith the detection or quantification of target analyte) in a sample andthereby allows a more accurate detection and diagnosis.

In one embodiment, the present method and device are applicable to anytypes of sample. In one embodiment, samples include but are not limitedto food, blood, plasma, serum, tissues, bacteria, viruses, RNA viruses,smear preparations, bacteria cultures, cell cultures (e.g. cellsuspensions and adherent cells), urine, saliva, fecal matters, andbodily discharges (e.g. tears, sputum, nasopharyngeal mucus, vaginaldischarge and penile discharge), polymerase chain reaction (PCR)mixtures and in vitro nucleic acid modification reaction mixtures.

In one embodiment, the target analytes include but are not limited toproteins, nucleic acids, carbohydrates, lipids, bacteria, virus,pathogens, food allergen and nanoparticles and the like.

In one embodiment, the target analyte is a food allergen including butis not limited to vegetable protein and animal protein. Lupine proteinis more prefer in the invention.

In one embodiment, the target analyte is a nucleic acid of various types(e.g. DNA including cDNA, RNA including mRNA and rRNA), forms (e.g.single-stranded, double-stranded, coiled, as a plasmid, non-coding orcoding) and lengths (e.g. an oligonucleotide, a gene, a chromosome andgenomic DNA), originated from the subject or an exogenous agent or both.

In one embodiment, the target analyte is a protein which is a peptide ora polypeptide, including an intact protein molecule, a degraded proteinmolecule and digested fragments of a protein molecule. In oneembodiment, biomarkers include but are not limited to antigens,receptors and antibodies, originated from the subject or an exogenousagent or both.

In one embodiment, the target analyte is a small molecule such as ametabolite. In one embodiment, the metabolite is a disease-relatedmetabolite which is indicative of the presence or extent of a disease ora health condition. In one embodiment, the metabolite is a drug-relatedmetabolite such as a drug by-product of which the level changes in asubject body consuming the drug.

In one embodiment, the target analyte is originated from the subjecthimself or herself (e.g. molecules that are derived or released from anyorgans, tissues or cells of the subject), an exogenous source (e.g. apathogen such as virus or bacteria associated with a particulardisease), or a food allergen (protein) from food or drug taken by thesubject. S. mutans is the prefer bacteria in this invention.

In one embodiment, the target analyte is not normally found in healthysubject. In one embodiment, the target analyte is a molecule that isnormally found in a healthy subject but the level of which is indicativeof a particular disease or a health condition.

ATPS (Aqueous Two-Phase System) within Porous Material

In one embodiment, the present invention provides use of ATPS componentswithin a porous material. Various ATPS systems can be used in thepresent invention, including but are not limited to polymer-polymer(e.g. PEG-PVP), polymer-salt (e.g. PEG-salt), and micellar. Porousmaterial may be made of any suitable porous material which can absorband transfer liquid. Suitable porous materials for this inventioninclude but are not limited to hydrogel, fiberglass paper, cotton-basedpaper, other types of paper, polymer foams, cellulose foams, other typesof foams, rayon fabric, cotton fabric, other types of fabric, wood,stones, and any other materials that can absorb and transfer liquid.

In one embodiment, the present invention provides uses of ATPS (aqueoustwo phase system) for the isolation and/or concentration of a targetanalytes in a sample solution, wherein the phase separationcharacteristics are altered by one or more phase separation modifyingagents that improve the behaviors and performance of the ATPS, therebyinduces or promotes phase separation of the ATPS.

In one embodiment, the ATPS comprises a mixed phase solution comprisinga first phase solution and a second phase solution, wherein componentsof said first phase solution and components of said second phasesolution are embedded in said porous material at a concentration or aloading that is sufficient to undergo a phase separation as the mixedphase solution flows through the porous material. In one embodiment, thephase separation characteristics are altered by one or more phaseseparation modifying agents so as to induce, stabilize and/or promotephase separation of the ATPS.

In one embodiment, components of the first phase solution and/or thecomponents of said second phase solution of the ATPS are embedded in theporous material and then dehydrated prior to the addition of a samplecontaining the target analyte to said porous material.

In one embodiment, components of the first phase solutions and/or thecomponents of said second phase solution of the ATPS are combined with asample containing the target analyte to create a mixture prior to theaddition of said mixture to the porous material.

In one embodiment, some of the components of the first phase solutionand/or the components of the second phase solution of the ATPS areembedded in the porous material and then dehydrated, while the remainingcomponents of the first phase solutions and/or the components of thesecond phase solution are combined with a sample containing the targetanalyte to create a mixture prior to the addition of the mixture to theporous material.

In one embodiment, there is provided a two-component ATPS (aqueous twophase system) within a porous material for the concentration of one ormore target analytes and/or the purification of a sample solution. Thetarget analyte is in contact with the mixed phase solution comprising afirst phase solution and a second phase solution, and partitions intothe first phase solution, the second phase solution or the interface (orinterphase) between the first phase solution and the second phasesolution.

In one embodiment, there is provided a two-component ATPS (aqueoustwo-phase system) within a porous material for removing one or morecontaminants from a sample, thereby obtains a purified sample of thetarget analyte(s). In one embodiment, the one or more contaminants arein contact with the mixed phase solution comprising a first phasesolution and a second phase solution, and wherein the contaminantspartitions into the first phase solution, the second phase solution, orthe interface (or interphase) between the first phase solution and thesecond phase solution.

In one embodiment, the porous material and ATPS are selected so that thefirst phase solution flows through the porous matrix at a first rate andthe second phase solution flows through the porous matrix at a secondrate, wherein the first rate and the second rate are different.

In one embodiment, the porous material is commercially available ormanufactured in-house.

Phase Separation Behavior Modifying Agents

In one embodiment, the present method and device comprise a porousmaterial embedded with ATPS components and one or more phase separationbehavior modifying agents that modify the phase separation behavior ofthe ATPS and thereby inducing or promoting phase separation. In oneembodiment, the present phase separation behavior modifying agents arecapable of widening the range of concentrations of ATPS components thatare effective for phase separation.

In one embodiment as shown in FIG. 1 , it is further contemplated thatthe present method and device may use different types of phaseseparation behavior modifying agents at various combinations. In oneembodiment, it is further contemplated that the phase separationbehavior modifying agent(s) can be disbursed within the porous materialof the device at many different locations and at differentconcentrations within each location.

A binodal curve denotes the conditions (e.g. temperature andconcentrations of the phase components) at which two distinct phases maycoexist and hence shows the boundary of conditions under which phaseseparation will occur. Under the theory of binodal curve, any pointoutside the binodal curve represents a monophasic system (no phaseseparation), while any point inside the binodal curve represents adiphasic system (phase separation).

FIG. 2 shows phase separation diagrams (binodal curves in terms oftemperature and relative concentrations of components forming the firstand second phase of the ATPS) under various flow rates comparing theseparation behavior and characteristics of ATPS components with orwithout adding behavior modifying agent to the system. On the left panelwhere no phase separation behavior modifying agent is added to the ATPS(Panels A-C of FIG. 2 ), two points can be observed on the binodal curveat a given temperature (except the extremum of the binodal curve),denoting a range of relative concentrations of the two-phase componentsat which the solution will undergo a phase separation at thattemperature. A solution of a certain composition having a relativeconcentration outside the range will exist as one single phase. On theright panel where phase separation behavior modifying agent is added tothe ATPS (Panels D-F of FIG. 2 ), similarly two points can be observedon the binodal curve at a given temperature and concentration of themodifying agent (except the extremum of a binodal curve). In FIG. 2 ,the horizontal solid line (“tie line”) within the boundary of thebinodal curve denotes conditions under which the two phases exist inequilibrium with each other at a given temperature. All systems on aparticular tie line produce the same two phases at that giventemperature and the intersections of the tie line with the binodal curverepresent compositions of the two phases that exist in equilibrium witheach other at that temperature. The volume ratio of the ATPS solutioncan be determined by comparing the length of the point on the tie lineto the binodal curve on the left to the length of the point to thebinodal curve on the right (referred to as the “lever arm rule”). Forexample in Panel A of FIG. 2 , the volume ratio of the two phasesolutions is 1:5 when the relative concentration of ATPS components isn, while in Panel B of FIG. 2 the volume ratio is 5:1 when the relativeconcentration of ATPS components is 2n. In contrast, when the relativeconcentration of ATPS components is 3n falling outside the tie line, thetwo phase solutions will not undergo a phase separation and remain asone single phase (Panel C of FIG. 2 ). Accordingly, the partitioncoefficient of a target analyte is the same along the tie lineregardless of changes in the relative concentration of ATPS components.Since the yield of the target analyte recovered by ATPS depends on thepartition coefficient of the target analyte and the volume of the targetphase recovered, the yield will depend on the tie line and the positionof the ATPS on that tie line. Typically, the longer the tie line is, thehigher the yield will be. The phase diagrams on the right panel (PanelsD-F of FIG. 2 ) indicated that addition of phase separation behaviormodifying agent to the ATPS shifts the position of the binodal curvedownward and thus lengthening the tie line at any given temperature,thereby allowing phase separation to occur within a wider range ofrelative concentration of the two phase components, as well asincreasing the partition coefficient. The range encompassed by the twopoints on the binodal curve is always wider in the presence of modifyingagents regardless of the flow rate. Furthermore, in certain embodimentswhere the ATPS components and the phase separation behavior modifyingagents are dehydrated within the porous material, the phase separationvolume ratio becomes more stable with respect to flow rate andfluctuations in the concentration of the ATPS components. Along the sametie line, an increase in ATPS component concentration will result inmore extreme volume ratios. Under the theory of binodal curve, if theoperating relative concentration of ATPS components is too close toeither side of the binodal curve, even a slight change of volume ratioof the two phase solutions (e.g. a slight dilution or concentration ofthe solutions) can cause a shift of the operating relative concentrationto another side of the binodal curve where only one phase exists.However, in cases where the porous material is embedded with both theATPS components and modifying agents in dehydrated form, the increase inATPS component concentration also correlates with an increase in phaseseparation behavior modifying agents, and therefore leading to aproportional downward shift of the binodal curve and a longer tie lineat temperature T as compared to the phase diagrams in Panels A-C of FIG.2 . As such, the same relative concentration of ATPS components wouldhave a smaller influence on the volume ratio when modifying agents arepresent, making the system more stable and robust. It should be notedthat FIG. 2 is illustrative of a temperature dependent ATPS with asingle ATPS component, however the concept described above similarlyapplies to ATPSs that are not temperature dependent and ATPSs that havemultiple ATPS components.

The binodal curves theory displayed in FIG. 2 is generally applicable toall type of modifying agents, including modifying agents describedherein. It is believed that the phase separation behavior modifyingagents are capable of altering the characteristics such as temperature,pH, hydrophobicity/hydrophilicity, surface tension, and/or ionicstrength of one of the phase components or both and allowing phaseseparation to occur within a wider range of relative concentration ofthe two-phase components, thereby triggering or promoting phaseseparation of ATPS.

In one embodiment, addition of phase separation behavior modifyingagents to ATPS components within a porous material improves theseparation behavior and performance characteristics of the ATPScomponents, including but not limited to increasing the stability orreducing fluctuations of ATPS through adjustment of parameters includingbut are not limited to total volume of a sample solution that undergoesphase separation, volume ratio, fluid flow rates and concentration ofATPS components.

In one embodiment, the present method and device comprise a porousmaterial embedded with ATPS components and one or more phase separationbehavior modifying agents to increase the yield of purified targetanalytes.

In one embodiment, the present method and device comprise a porousmaterial embedded with ATPS components and one or more phase separationbehavior modifying agents that improve the stability of ATPS duringphase separation by minimizing fluctuations in the ATPS.

In one embodiment, the method and device comprise a porous materialembedded with ATPS components and one or more phase separation behaviormodifying agents to remove contaminants from a sample solution.

In one embodiment, the phase separation behavior modifying agent(s)is/are combined with a sample containing the target analyte, and/or oneor more ATPS components to create a mixture prior to the addition ofsaid mixture to the porous material. In one embodiment, the phaseseparation behavior modifying agent(s) is/are combined with a samplecontaining the target analyte, and/or one or more ATPS components in asequential order prior to the addition of each to the porous material.

In one embodiment, the phase separation behavior modifying agent(s)is/are embedded in the porous material containing ATPS components andthen dehydrated prior to the addition of a sample to the porousmaterial.

In one embodiment, some of the phase separation behavior modifyingagent(s) is/are embedded in the porous material containing ATPScomponents and then dehydrated, while the remaining phase separationbehavior modifying agent(s) is/are combined with a sample containing thetarget analyte to create a mixture prior to the addition of the mixtureto the porous material.

In one embodiment, some of the phase separation behavior modifyingagent(s) is/are embedded in the porous material containing components ofeither the first or second phase solution of the ATPS and thendehydrated, while the remaining phase separation behavior modifyingagent(s) is/are combined with components of the other phase solution ofthe ATPS to create a mixture prior to the addition of said mixture tothe porous material.

In one embodiment, some of the phase separation behavior modifyingagent(s) is/are embedded on the porous material without ATPS componentsand then dehydrated, while the remaining phase separation behaviormodifying agent(s) is/are combined with components of the first phasesolution of the ATPS and components of the second phase solution of theATPS to create a mixture prior to the addition of said mixture to theporous material.

In one embodiment, the phase separation behavior modifying agent(s)is/are present in a dry form in some regions of the porous material. Inone embodiment, the phase separation behavior modifying agent(s) is/areembedded in some regions of the porous material. In another embodiment,the phase separation behavior modifying agent(s) is/are embedded in theentire region of the porous material.

In one embodiment, the phase separation behavior modifying agent(s)is/are present in a dry form at a uniform concentration across all theregions of the porous material. In another embodiment, the phaseseparation behavior modifying agent(s) is/are present in a dry form atvarying concentrations in different regions of the porous material.

In one embodiment, the present separation behavior modifying agentsinclude but are not limited to:

-   -   a. compounds carrying an acidic functional group in aqueous        solution, selected from dextran, sucrose, saccharides,        polysaccharides, sorbates, polysorbates, carboxylates,        polycarboxylates, phosphates, potassium phosphate,        polyphosphates, sulfates and polysulfates, polyols;    -   b. compounds carrying an amine functional group in aqueous        solution, selected from amines, polyamines, amine salts and        polyamine salts;    -   c. compounds carrying both a hydrophilic group and a hydrophobic        group, selected from lipids, surfactants, nucleic acids,        hormones, proteins, amino acids;    -   d. chaotropic agents; and    -   e. kosmotropic agents.

In one embodiment, the present phase separation behavior modifyingagents include, but are not limited to, sucrose, potassium phosphates,dextran, Tween-20 and Triton-X114.

Adjusting or Stabilizing the Total Volume of Either Phase or Both Phases

In one embodiment, the present phase separation behavior modifyingagent(s) is/are selected to:

-   -   a) reduce fluctuations (increase stability) in the total volume        of either or both phases during and after phase separation. For        example, when the ATPS components are embedded and dehydrated        within a porous material and resolubilized during fluid flow,        continued resolubilization of the ATPS components will change        the total volumes of the first phase and/or the second phase.        Variability in flow rates results in variability in the total        volumes of the first phase and/or the second phase. In one        embodiment, a modifying agent such as compound carrying both        hydrophilic and hydrophobic group is used, the compound        interacts with the dehydrated ATPS components within the porous        material and hence reduce the fluctuations of total volume of        the first phase and/or the second phase during the flow of fluid        within the porous material. Reduced fluctuations in the fold of        concentration is desirable for more robust devices; or    -   b) increase the total volume of a sample solution that undergoes        phase separation. When the ATPS components are dehydrated within        a porous material and resolubilized during fluid flow, continued        resolubilization of the ATPS components will increase the total        volume of the first phase and/or the second phase. In one        embodiment, a compound carrying acidic functional group in        aqueous solution is used as a modifying agent to decrease the pH        value of the first phase and/or the second phase solution and        thereby promoting resolubilization of the first phase and/or the        second phase solution and increasing the total volume of the        phase solution. Overall, the compound carrying acidic function        group will cause phase separation to occur earlier and/or at a        lower concentration of ATPS within a porous material, this will        result in a larger volume of the fluid undergoing phase        separation as it flows through the porous material. This may be        beneficial to concentrate more target from a given solution in        the phase of interest; or    -   c) decrease the total volume of a sample solution that undergoes        phase separation. In one embodiment, a compound carrying amine        functional group in aqueous solution is used as a modifying        agent to increase the pH value of the first phase and the second        phase and thereby hindering the resolubilization of the two        phases. When the ATPS components are dehydrated within a porous        material and resolubilized during fluid flow, the hindered        resolubilization of the ATPS components will decrease the total        volume of the first phase and/or the second phase. Therefore,        phase will separate later and/or at a higher concentration of        ATPS components within a porous material, this will decrease the        total volume of the fluid undergoing phase separation as it        flows through the material. This may be beneficial to        concentrate more target from a given solution in the phase of        interest.

Adjusting or Stabilizing the Volume Ratio of the First and the SecondPhase

In one embodiment, the present phase separation behavior modifyingagent(s) is/are selected to:

-   -   a) reduce fluctuations (increase stability) in the volume ratio        between the first and the second phase of the ATPS during or        after the course of phase separation. When the ATPS components        are dehydrated within a porous material and resolubilized when        the fluid flows within the porous material, continued        resolubilization of the ATPS components will change the volume        ratio of the two phases. Variability in flow rates results in        variability in volume ratio. In one embodiment, by adding a        modifying agent such as compound carrying both hydrophilic and        hydrophobic groups, the agent will interact with the dehydrated        ATPS components by hydrophilic and hydrophobic groups and reduce        the fluctuations in the volume ratio when the fluid flows within        the porous material. Reduced fluctuations in volume ratio is        desirable for more robust devices; or    -   b) increase the volume ratio between the first and the second        phase of the ATPS. In one embodiment, by adding a modifying        agent such as compound carrying acidic functional group in        aqueous solution to an ATPS (depending on which phase is the        phase of interest), the pH value in the phase of interest will        be decreased and thereby promoting the resolubilization of that        phase of interest. An increase in volume ratio may be useful to        further increase target concentration, to improve the flow of        fluid, or to desirably adjust the volume of the phase of        interest. This may be beneficial to concentrate more target from        a given solution in the phase of interest; or    -   c) decrease the volume ratio between the first and the second        phase of the ATPS. In one embodiment, by adding a modifying        agent such as a compound carrying amine functional group in        aqueous solution to an ATPS (depending on which phase is the        phase of interest), the pH value in the phase of interest will        be increased and thereby hindering the resolubilization of that        phase of interest. A decrease in volume ratio may be useful to        further increase target concentration, to improve the flow of        fluid, or to desirably adjust the volume of the fluid phase of        interest. This may be beneficial to concentrate more target from        a given solution in the phase of interest.

Adjusting the Concentrations of the First and the Second PhaseComponents

In one embodiment, the present phase separation behavior modifyingagent(s) is/are selected to:

-   -   a) reduce fluctuations in the concentrations of the first phase        component and/or the second phase component within the first        phase or within the second phase. When the ATPS components are        embedded on and dehydrated within a porous material and        resolubilized during fluid flow, continued resolubilization of        the ATPS component will change the concentrations of the first        phase components and/or the second phase components. Variability        in flow rates results in variability in the concentrations of        components in the first phase and/or second phase. In one        embodiment, by adding a modifying agent such as a chemical        carrying both hydrophilic and hydrophobic groups into the        solution or the porous material, the agent will interact with        ATPS components by hydrophilic and hydrophobic groups and reduce        the fluctuations in the concentration when the fluid flows        within the porous material. Reduced fluctuations in the fold of        concentration is desirable for more robust devices; or    -   b) increase the concentration of components of one phase of the        ATPS within the same phase. In one embodiment, when the ATPS        components are dehydrated within the porous material, by either        adding a modifying agent such as a chemical carrying acidic        functional group in aqueous solution directly to the solution        containing the target analyte or dehydrating the modifying agent        within the porous material, the pH value in that phase solution        will be will decreased and thereby promoting resolubilization of        the ATPS components in that phase, resulting in a higher        concentration. This may be beneficial to increase the        concentration factor and thus enhance the concentration of the        target analyte by increasing the volume ratio which causes more        volume to undergo phase separation. This may be beneficial to        concentrate more target from a given solution in the phase of        interest; or    -   c) decrease the concentration of components of one phase of the        ATPS within the same phase. In one embodiment, by either adding        a modifying agent such as a compound carrying amine functional        group in aqueous solution directly to the solution containing        the target analyte or dehydrating the modifying agent within the        porous material, it will increase the pH value and reduce the        rate of resolubilization of ATPS components of that phase, thus        resulting in a lower concentration of ATPS components of one        phase within the same phase during resolubilization. This may be        beneficial to create a more sustained release of ATPS components        as the fluid flows within the porous material, resulting in a        more uniform ATPS concentration and a larger volume that        undergoes phase separation. This may be beneficial to        concentrate more target from a given solution in the phase of        interest; or    -   d) increase the concentration of the components of one phase        within the other phase. In one embodiment, when the ATPS        components are dehydrated within the porous material, by either        adding a compound carrying acidic functional group in aqueous        solution directly to the solution containing the target analyte        or dehydrating the modifying agent within the porous material,        the pH value of the first phase components within the second        phase will be decreased and thereby promoting resolubilization        of components of the first phase within the second phase        solution, resulting in a higher concentration of the first phase        components within the second phase. This may be beneficial to        increase the volume ratio, cause more volume to undergo phase        separation, and/or increase the concentration of the target        analyte. This may be beneficial to concentrate more target from        a given solution in the phase of interest; or    -   e) decrease the concentration of components of one phase of the        ATPS within the other phase. In one embodiment, by either adding        a modifying agent such as a compound carrying amine functional        group in aqueous solution directly to the solution containing        the target analyte or dehydrating the modifying agent within the        porous material, the pH value and the rate of resolubilization        of components of that phase will be increased, thus resulting in        a lower concentration of ATPS components of one phase within the        other phase during resolubilization. This may be beneficial to        create a more sustained release of ATPS component as the fluid        flows within the porous material, resulting in more uniform ATPS        concentration and a larger volume that undergoes phase        separation. This may be beneficial to concentrate more target        from a given solution in the phase of interest; or    -   f) any combination of a-e.        Inducing Phase Separation at an Earlier Time and/or at a Lower        Concentration of ATPS Components within a Porous Material

In one embodiment, by causing the phase separation to occur earlierand/or at a lower concentration of ATPS components within a porousmaterial, this will result in a larger volume of the fluid undergoingphase separation as it flows through the porous material. In oneembodiment, this can be achieved by adding a modifying agent such as acompound carrying acidic functional group in aqueous solution todecrease the pH value of the first and/or second phase and therebypromoting the resolubilization of the phase components. When the ATPScomponents are dehydrated within a porous material and resolubilized asthe fluid flows, continued resolubilization of the ATPS components willincrease the total volumes of the first phase and/or the second phase.This may be beneficial to concentrate more target from a given solution.In one embodiment, the phase separation behavior modifying agent(s)is/are selected to induce initial phase separation at a lowerconcentration of the ATPS components that would otherwise be too low toinduce phase separation in the absence of said phase separation behaviormodifying agent(s). In one embodiment where the ATPS comprises amicellar solution, when the concentration of ATPS components isinsufficient to cause a phase separation, one or more modifying agentscan be added to alter parameters of the ATPS components such astemperature, pH value or ionic strength to allow phase separation at alower concentration. This would benefit situations where the temperatureis too low to run the diagnostic, i.e., by expanding the operatingtemperature range, or situations where a downstream diagnostic componentwas sensitive to the ATPS components which requires their concentrationsbe kept at low level.

In one embodiment, by causing the phase separation to occur earlierand/or at a lower concentration of ATPS components within a porousmaterial, it will result in a larger volume of the fluid undergoingphase separation as it flows through the porous material. In oneembodiment, this can be achieved by adding a modifying agent such as acompound carrying acidic functional group in aqueous solution todecrease the pH value of the first phase and/or second phase and therebypromoting the resolubilization of the components. When the ATPScomponents and/or modifying agents are dehydrated within a porousmaterial and resolubilized as the fluid flows, continuedresolubilization of the ATPS component will increase the total volume ofthe first phase and/or the second phase. This may be beneficial toconcentrate more target analyte from a given solution. In situationswhere the ATPS components are dehydrated within the porous material andrequire resolubilization, phase separation will only occur once theconcentration has reached a certain point. By adding a modifying agentthat induces phase separation at a lower concentration of the ATPScomponents, phase separation will start earlier at a more upstreamposition within the porous material. This may be beneficial to increasethe total volume that undergoes phase separation and thus concentratemore target analyte for the downstream application.

In one embodiment as shown in FIG. 1 , the present method and/or devicemay use many different types and many variations of combinations ofphase separation behavior modifying agents. In one embodiment, the phaseseparation behavior modifying agent(s) may be distributed within theporous material of the device at multiple different locations and/or atdifferent concentrations within each location.

Adjusting the Rate of Domain Coalescence of the ATPS

Domain coalescence is the process where small drops of one phase of anATPS coalescing together to form a larger volume of that phase. In oneembodiment, the present phase separation behavior modifying agent(s)is/are selected to:

-   -   a) reduce fluctuations in the rate of domain coalescence of one        or both phases of an ATPS. In one embodiment, by adding charged        surfactant or protein that are self-interacting to the solution,        these agents will partition to the interface between the domains        of one of the phases and increase domain to domain interaction,        thereby reducing the fluctuations in the rate of domain        coalescence; or    -   b) increase the rate of domain coalescence of one or both phases        of an ATPS. In one embodiment, by adding charged surfactant or        protein that are self-interacting to the solution, these agents        will partition to the interface between the domains of one of        the phases and increase domain to domain interaction, thus        increasing the rate of domain coalescence. This may be        beneficial to speed up the run time of the diagnostic; or    -   c) decrease the rate of domain coalescence of one or both phases        of an ATPS. In one embodiment, by adding surfactant to the        solution which decreases the surface tension between the two        phases. With decreased surface tension, there is less of a        thermodynamic force to drive domain coalescence. This may be        beneficial in situations where the target analyte partition        slowly and hence requires more time to get to the appropriate        phase which is facilitated by keeping domain small initially by        slowing down their coalescence;

Adjusting the Flow Rate of Fluids

In one embodiment, the flow rate within a porous material can beincreased or decreased by adding agents that change the viscosity and/orhydrophobicity of the solution. Generally, flow rate of fluid isinversely proportional to its viscosity. Flow rate also increases as thehydrophobicity of the fluid becomes more similar to the porous material.In one embodiment, the present phase separation behavior modifyingagent(s) is/are selected to:

-   -   a) reduce fluctuations in the flow rate within the porous matrix        of the first phase, or the second phase. In one embodiment, by        adding a modifying agent such as compound carrying both        hydrophilic and hydrophobic groups, the agent will interact with        ATPS components by hydrophilic and hydrophobic groups and reduce        the fluctuations in flow rate; or    -   b) increase the flow rate within the porous matrix of the first        phase or the second phase. In one embodiment, by adding a        modifying agent such as a compound carrying hydrophilic groups,        the agent will interact with ATPS components by hydrophilic        groups to decrease the ionic strength of the system and as a        result, decrease the viscosity and hydrophobicity of the        solution and the flow rate will increase; or    -   c) decrease the flow rate within the porous matrix of the first        phase or the second phase. In one embodiment, by adding a        modifying agent, such as a compound carrying hydrophobic groups,        the agent will interact with ATPS components by hydrophobic        groups to increase the ionic strength of the system. As a        result, the viscosity and hydrophobicity of the solution are        increased and the flow rate will decrease; or    -   d) any combination of a-c.        Adjusting the Partitions of Target Analyte or Contaminants into        the First or Second Phase Components

In one embodiment, the present phase separation behavior modifyingagent(s) is/are selected to:

-   -   a) reduce fluctuations in the partitioning of target analyte        and/or contaminants between the first phase and the second        phase; or    -   b) increase the partition of the target analyte and/or        contaminants to the phase of interest. Target partitioning can        be driven by a variety of forces/principles including without        limitation size exclusion principles, hydrophobic/hydrophilic        interactions, electrostatic interactions. In one embodiment, by        adding modifying agent that changes the environment within a        phase, partitioning of the target analyte to the phase of        interest can be increased. For example, the addition of        chaotropic agents will make the hydrophobic phase more        hydrophobic by disrupting the hydrogen bonds in the interested        phase, thus increasing the partitioning of hydrophobic analyte        or contaminants to that phase. This may be beneficial to further        increase concentration of the target analyte or contaminants; or    -   c) decrease the partition of the target analyte and/or        contaminants to the phase of interest. In one embodiment, adding        kosmotropic agents will make the hydrophobic phase more        hydrophilic by increasing the interactions between the two        phases, thus decreasing the partitioning of hydrophobic analyte        or contaminants to that phase. This may be desirable if one is        trying to separate two targets into different phases; or    -   d) any combination of a-c.

In one embodiment, the phase separation behavior modifying agent(s)is/are selected to perform any combination of functions described above.

Adjustment of Concentration Factors

In one embodiment, the relative amounts of ATPS components in the porousmaterial can be changed.

In one embodiment, by changing the amounts of ATPS components dehydratedon the porous material and thereby the volume ratio of the two phases,the target analyte can be preferentially concentrated in one phase. Inone embodiment, the target analyte is retained in the leading front ofthe ATPS, which is then collected and optionally further analyzed usingappropriate technologies.

In one embodiment, the order of ATPS components on the porous materialcan be adjusted to achieve the desired effect or phenomenon. In oneembodiment, one or more ATPS components can be dehydrated and embeddedin the porous material.

In one embodiment, to better quantify the phenomena associated with thepresent invention, an assay was developed to evaluate the correlationbetween the relative amounts of ATPS components dehydrated on the porousmaterial and the fold of concentration achieved. In one embodiment, theconcentration factor can be controlled and fine-tuned by adjusting therelative amount of the ATPS components as needed.

In one embodiment, to integrate the ATPS components into the porousmaterial, the ATPS components were solubilized in water (or appropriatebuffer) and applied a predetermined amount of the solution/suspension onthe porous material. The porous materials were then placed in alyophilizer or equivalent solvent-removing equipment to remove solvent(e.g., water), resulting in dehydration of the ATPS components directlyon the porous material. Upon introduction of the sample solution to theporous material, the ATPS components instantly undergo rehydration andthereby separating the components or molecules in the sample as theyflow within the porous material and concentrating the target analyte atthe front of the fluid flow. In one embodiment, no external power orequipment is required to provide a driving force.

In one embodiment, the present invention provides a device comprising aporous fiberglass paper that is impregnated with a polymer/polymer ATPS,e.g., PEG-PVP ATPS. When a sample containing a plurality of analytes isapplied to one end of the device, the impregnated porous fiberglasspaper causes the analyte-containing ATPS component to preferentiallyflow ahead of the other ATPS components. Therefore, the target analyteis concentrated in the analyte-containing ATPS component at the front ofthe fluid flow.

In one embodiment, the present invention provides a device comprising aporous fiberglass paper that is pretreated with a polymer-salt ATPS,e.g., PEG-salt ATPS. When a sample containing a plurality of analytes isapplied to one end of the device, the pretreated porous fiberglass papercauses the analyte-containing ATPS component to preferentially flowahead of the other ATPS components. Therefore, the target analyte isconcentrated in the analyte-containing ATPS component at the front ofthe fluid flow.

In one embodiment, the present invention provides a device comprising aporous fiberglass paper is impregnated with a micellar ATPS. When asample containing a plurality of analytes is applied to one end of thedevice, the impregnated porous fiberglass paper causes theanalyte-containing ATPS component to preferentially flow ahead of theother ATPS component. Therefore, the target analyte is concentrated inthe analyte-containing ATPS component at the front of the fluid flow.

In one embodiment, the sample solution suitable for the device/methoddisclosed in the present invention is a buffer solution, which includes,but is not limited to, phosphate-buffered saline (PBS), and Tris-EDTA(TE) buffer. In this invention, preferably, the buffer solution is PBSbuffer (8 mM Sodium chloride, 0.2 mM Potassium chloride, 1.15 mM Sodiummonohydrogen phosphate, 0.2 mM Potassium dihydrogen phosphate solutionwith pH 7.35-7.65). In one embodiment, the buffer solution is TE buffercontaining 2% bovine serum albumin (BSA), 0.1% PEG and 20 mM Tris, pH7.5 respectively.

In one embodiment, multiple ATPS components are incorporated into aporous material to achieve a control of concentration factors of thetarget analyte.

In one embodiment, the optimized ratios of the ATPS components areimplemented in a polymer/salt ATPS, polymer/polymer ATPS, or micellarATPS to control the concentration factors.

In one embodiment, the ratios of the ATPS components are adjusted todemonstrate concentration factors between 10 folds and 100 folds.

In one embodiment, there are various ATPS systems including but notlimited to polymer-polymer (e.g., PEG-PVP), polymer-salt (e.g.PEG-salt), and micellar. The first and/or second component comprises apolymer. Polymer includes but is not limited to polyalkylene glycols,such as hydrophobically modified polyalkylene glycols,poly(oxyalkylene)polymers, poly(oxyalkylene)copolymers, such ashydrophobically modified poly(oxyalkylene)copolymers, polyvinylpyrrolidone (PVP), polyvinyl alcohol, polyvinyl caprolactam, polyvinylmethylether, alkoxylated starches, alkoxylated cellulose, alkylhydroxyalkyl cellulose, silicone-modified polyethers, and polyN-isopropylacrylamide and copolymers thereof. In another embodiment, thefirst polymer comprises polyethylene glycol, polypropylene glycol.

In one embodiment, the polymer concentration of the first or secondphase is in the range of about 0.01% to about 90% by weight of the totalweight of the aqueous solution (w/w). In various embodiments, thepolymer solution is selected from a polymer solution that is about 0.01%w/w, about 0.05% w/w, about 0.1% w/w, about 0.15% w/w, about 0.2% w/w,about 0.25% w/w, about 0.3% w/w, about 0.35% w/w, about 0.4% w/w, about0.45% w/w, about 0.5% w/w, about 0.55% w/w, about 0.6% w/w, about 0.65%w/w, about 0.7% w/w, about 0.75% w/w, about 0.8% w/w, about 0.85% w/w,about 0.9%) w/w, about 0.95% w/w, or about 1% w/w. In some embodiments,the polymer solution is selected from polymer solution that is about 1%w/w, about 2% w/w, about 3% w/w, about 4% w/w, about 5% w/w, about 6%w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11%w/w, about 12% w/w, about 13% w/w, about 14% w/w, about 15% w/w, about16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, about 20% w/w,about 21% w/w, about 22% w/w, about 23% w/w, about 24% w/w, about 25%w/w, about 26% w/w, about 27% w/w, about 28% w/w, about 29% w/w, about30% w/w, about 31% w/w, about 32% w/w, about 33% w/w, about 34% w/w,about 35% w/w, about 36% w/w, about 37% w/w, about 38% w/w, about 39%w/w, about 40% w/w, about 41% w/w, about 42% w/w, about 43% w/w, about44% w/w, about 45% w/w, about 46% w/w, about 47% w/w, about 48% w/w,about 49% w/w, and about 50% w/w.

In one embodiment, the first and/or second phase comprises a salt, thesalt includes but is not limited to, inorganic salts containing cationssuch as straight or branched trimethyl ammonium, triethyl ammonium,tripropyl ammonium, tributyl ammonium, tetramethyl ammonium, tetraethylammonium, tetrapropyl ammonium and tetrabutyl ammonium, and anions suchas sulphate, nitrate, chloride and hydrogen carbonate. In anotherembodiment, the salt is selected from the group consisting of NaCl,Na₂SO₄, potassium citrate, (NH₄)₂SO₄, sodium citrate, sodium acetate,sodium cholate and combinations thereof. Other salts, e.g. ammoniumacetate, may also be used.

In one embodiment, the total salt concentration is in the range of 0.001mM to 100 mM. A skilled person in the art will understand that theamount of salt needed to form an aqueous two-phase system will beinfluenced by molecular weight, concentration and physical status of thepolymer.

In various embodiments, the salt phase is selected from a salt solutionthat is about 0.001% to 90% w/w. In various embodiments, the saltsolution is selected from a salt solution that is about 0.01% w/w, about0.05% w/w, about 0.1% w/w, about 0.15% w/w, about 0.2% w/w, about 0.25%w/w, about 0.3% w/w, about 0.35% w/w, about 0.4% w/w, about 0.45% w/w,about 0.5% w/w, about 0.55% w/w, about 0.6% w/w, about 0.65% w/w, about0.7% w/w, about 0.75% w/w, about 0.8% w/w, about 0.85% w/w, about 0.9%)w/w, about 0.95% w/w, or about 1% w/w. In some embodiments, the saltsolution is selected from polymer solution that is about 1% w/w, about2% w/w, about 3% w/w, about 4% w/w, about 5% w/w, about 6% w/w, about 7%w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12%w/w, about 13% w/w, about 14% w/w, about 15% w/w, about 16% w/w, about17% w/w, about 18% w/w, about 19% w/w, about 20% w/w, about 21% w/w,about 22% w/w, about 23% w/w, about 24% w/w, about 25% w/w, about 26%w/w, about 27% w/w, about 28% w/w, about 29% w/w, about 30% w/w, about31% w/w, about 32% w/w, about 33% w/w, about 34% w/w, about 35% w/w,about 36% w/w, about 37% w/w, about 38% w/w, about 39% w/w, about 40%w/w, about 41% w/w, about 42% w/w, about 43% w/w, about 44% w/w, about45% w/w, about 46% w/w, about 47% w/w, about 48% w/w, about 49% w/w, andabout 50% w/w.

In one embodiment, the first and/or the second phase in the ATPScomprises a solvent that is immiscible with water. In some embodiments,the solvent comprises a non-polar organic solvent. In some embodiments,the solvent comprises an oil. In some embodiments, the solvent isselected from pentane, cyclopentane, benzene, 1,4-dioxane, diethylether, dichloromethane, chloroform, toluene and hexane.

In one embodiment, the first phase and/or second phase in the ATPScomprises a micellar solution.

In one embodiment, one phase in the ATPS comprises a micellar solutionand the other phase comprises a liquid phase comprising a polymer. Inone embodiment, one phase in the liquid phase comprises a micellarsolution and the other phase in the liquid phase comprises a saltsolution. In one embodiment, one phase comprises a first polymer and theother comprises a second polymer. In one embodiment, the first/secondpolymer is selected from polyethylene glycol and PVP. In one embodiment,one phase comprises a polymer and the other phase comprises a salt. Insome embodiments, one phase comprises PVP and the other phase comprisessodium cholate. In one embodiment, one phase comprises a salt and theother comprises another salt.

In one embodiments, the volume ratios of the first phase to the secondphase are in the range of 1:1 to 1:1000. In some embodiments, the ratioof the first phase to the second phase is selected from a ratio of about1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7,about 1:8, about 1:9, and about 1:10. In some embodiments the ratio ofthe first phase to the second phase is selected from a ratio of about1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about1:80, about 1:90, and about 1:100. In some embodiments the ratio of thefirst phase to the second phase is selected from a ratio of about 1:200,about 1:300, about 1:400, about 1:500, about 1:600, about 1:700, about1:800, about 1:900, and about 1:1000.

In one embodiment, the volume ratios of the second phase to the firstphase are selected from a ratio of about 1:1, about 1:2, about 1:3,about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, andabout 1:10. In some embodiments the ratio of the second phase to thefirst phase is selected from a ratio of about 1:20, about 1:30, about1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, andabout 1:100. In some embodiments the ratio of the second phase to thefirst phase is selected from a ratio of about 1:200, about 1:300, about1:400, about 1:500, about 1:600, about 1:700, about 1:800, about 1:900,and about 1:1000.

Downstream Processing of the Isolated Analyte

In various embodiments, the present invention can be integrated with asecond device at the downstream region of the porous material of saidfirst device, such that the leading fluid within the porous materialwould transfer into said second device.

In various embodiments, the present invention can be integrated with asecond device at the downstream region of the porous material of saidfirst device, such that the leading fluid within the porous materialwould transfer into said second device. The phase separation behaviormodifying agent(s) is/are selected to benefit either directly orindirectly the performance of said second device integrated at thedownstream region through any combination of means described in thisinvention.

Analytes and/or analytes-containing solution obtained by the presentinvention can be used in conjugation with a wide range of downstreamapplications such as detection or analysis of the analytes in forensic,diagnostic or therapeutic applications, and laboratory procedures suchas sequencing, amplification, reverse transcription, labeling,digestion, blotting procedures and the like. The present invention canimprove the performance of downstream characterization or processing ofthe analytes.

In one embodiment, downstream applications of the analyte include, butare not limited to, any detection, analytical or diagnostic proceduresinvolving the detection or quantification of the purified analyte. Inone embodiment, detection, analytical or diagnostic procedures to becoupled with the present invention include but are not limited to anysuch procedure performed in commercial clinical laboratory, andlaboratory procedures such as sequencing, amplification (e.g. PCR,RT-PCR, real-time PCR, and real-time RT-PCR), reverse transcription,labeling, digestion, blotting procedures, ELISA, RIA, immunoassays,enzymatic assays, GC/MS, proteomic-based approach, and the like.

In one embodiment, analyte and/or analytes-containing solution obtainedby the present invention can be analyzed by a lateral flow assay (LFA).LFA has a number of desirable characteristics including their ease ofuse and board applicability to a variety of analytes. However, LFA isgenerally only capable of providing qualitative results due to itsdetection limitation. For example, the detection limit of LFA onpathogen (S. mutans) is 10⁶ CFU/ml. In the present invention whereinmodifying agent is used to improve the phase separation of the ATPS, thedetection limit of LFA on S. mutans can be improved to as low as 10⁴CFU/ml amounting to a 100-fold enhancement. Together with the improvedconcentration fold by 100-fold, the present invention is suited forproviding quantitative result in a broader range.

In one embodiment, the partition of the target analyte to the phase ofinterest can be enhanced by adding modifying agent to the ATPS. In oneembodiment, the fold of concentration is improved by 100-fold. Theimproved partition of the target analyte is also beneficial to increaseyield of target analyte.

It may produce a false negative result if the target analyte hasextremely low concentration. In one embodiment, due to the increasedconcentration of the target analyte by adding one or more modifyingagent(s) to the ATPS, the detection limit of LFA is improved. As aresult, the reproducibility of the test is increased.

In one embodiment, by adding one or more modifying agent(s), phaseseparation of the ATPS can be induced to occur earlier and/or at a lowerconcentration of the ATPS components. As a result, efficiency or speedof the entire detection or diagnostic process can be increasedsignificantly.

In various embodiments, the present invention can be used in combinationwith one or more processes or reagents for the purpose of washing andelution of the analytes retained in the porous matrix, or post-isolationtreatment of the retained analytes.

The first or second device is designed to enable manual and/or automatedextraction of the first phase solution and/or the second phase solutionfrom the porous material of said first or second device, and theextracted solution is then used for another method or process.

In various embodiments, the present phase separation behavior modifyingagent(s) is/are selected to benefit either directly or indirectly theperformance of said method or process that uses said extracted phasethrough any combination of means described in this invention.

In one embodiment, after contacting the analytes containing solutionwith the ATPS, a washing buffer is applied on the ATPS, once or formultiple times, to wash off non-target analytes or impurities from theporous matrix. Washing buffers may comprise solutions of varying ionicstrength, pH values, or contain additives such as detergents. Washingbuffers include but are not limited to a solution of 20%-50% ethanol and20%-50% isopropanol; a solution of about 0.1-4.0 M guanidiniumhydrochloride, detergents and up to about 80% ethanol; or a solution ofabout 80% ethanol.

In one embodiment, target analytes enter the porous matrix of the ATPSand flow from one end to the other end. In one embodiment, targetanalytes isolated by the present invention are eluted out of the porousmatrix using appropriate elution buffers or deionized water. In oneembodiment, isolated target analytes are not eluted but stored in theporous matrix for future use. For instance, after the isolation ofanalytes using the present invention, the porous material (e.g., apaper) containing the target analytes (e.g., a DNA) is dried and stored.In one embodiment, analytes retained on the porous matrix can be elutedfor further analysis or treatment. The selection of the elution buffermay depend on the contemplated use of the purified biomarker. Examplesof suitable elution buffers for nucleic acid type of analytes includes,but are not limited to, Tris-EDTA (TE) buffer, aqua bidest and PCRbuffer. In one embodiment, the purified analyte on porous paper iseluted in a tube containing TE buffer (10 mM TrisCl, 1 mM EDTA solutionwith pH 7.5), and the purified analyte is recovered in a relativelysmall volume, e.g., less than 100 μl.

In one embodiment, the present invention provides a device for isolatingor concentrating one or more target analytes, the device comprising:

-   -   i) a porous material;    -   ii) components capable of forming a first phase solution and a        second phase solution of an aqueous two-phase system (ATPS); and    -   iii) one or more phase separation behavior modifying agents;

wherein said components and said phase separation behavior modifyingagents are embedded in said porous material, wherein when a solutioncontaining said target analytes flows through said porous material, anaqueous two-phase system comprising said first phase solution and saidsecond phase solution is generated, wherein the separation behavior ofthe two phase solutions is altered by said phase separation behaviormodifying agents, and said target analytes are isolated or concentratedon said porous material, or

wherein one or more of said components and said phase separationbehavior modifying agents are embedded in said porous material, whereinthe balance of said components and said phase separation behaviormodifying agents are to be mixed with a solution containing said targetanalytes, thereby forming a mixed solution, wherein when said mixedsolution flows through said porous material, an aqueous two-phase systemcomprising said first phase solution and said second phase solution isgenerated, wherein the separation behavior of the two phase solutions isaltered by said phase separation behavior modifying agents, and saidtarget analytes are isolated or concentrated on said porous material.

In one embodiment, the one or more phase separation behavior modifyingagents are present in some regions of the porous material, and notpresent in other regions of the porous material.

In one embodiment, one or more phase separation behavior modifyingagents are evenly distributed on said porous material or present in anincreasing amount from one end to the other end of said porous material.

In one embodiment, one or more phase separation behavior modifyingagents are present in varying amounts in different regions of the porousmaterial.

In one embodiment, one or more target analytes partition into the firstphase solution or the second phase solution, or accumulate at aninterface between the first phase solution and the second phasesolution.

In one embodiment, the porous material and said components are selectedso that said first phase solution and second phase solution flow throughthe porous material at different rates.

In one embodiment, one or more phase separation behavior modifyingagents are selected from the group consisting of:

a) compounds carrying one or more acidic functional groups;

b) compounds carrying one or more amine functional groups; and

c) compounds carrying both hydrophilic and hydrophobic groups.

In one embodiment, the compounds carrying one or more acidic functionalgroups are selected from the group consisting of saccharides,polysaccharides, sorbates, polysorbates, carboxylates, polycarboxylates,phosphates, polyphosphates, sulfates, polysulfates and polyols.

In one embodiment, said compounds carrying one or more amine functionalgroups are selected from the group consisting of amines, polyamines,amine salts and polyamine salts.

In one embodiment, the compounds carrying both hydrophilic andhydrophobic groups are selected from the group consisting of lipids,surfactants, nucleic acids, hormones, proteins, and amino acids.

In one embodiment, the phase separation behavior modifying agent is achaotropic agent. In one embodiment, the phase separation behaviormodifying agent is a kosmotropic agent.

In one embodiment, one or more phase separation behavior modifyingagents are selected to:

-   -   a) reduce fluctuations in the total volume of the first phase        solution, the second phase solution, or both during or after        phase separation;    -   b) increase or decrease the total volume of the solution        containing said target analytes that undergoes a phase        separation;    -   c) reduce fluctuations in the volume ratio between the first        phase solution and the second phase solution; or    -   d) increase or decrease the volume ratio between the first phase        solution and the second phase solution.

In one embodiment, one or more phase separation behavior modifyingagents are selected to:

-   -   a) reduce fluctuations in the concentrations of one or more        components within the first phase solution;    -   b) reduce fluctuations in the concentrations of one or more        components within the second phase solution;    -   c) increase or decrease the concentration of one or more        components within the first phase solution; or    -   d) increase or decrease the concentration of one or more        components within the second phase solution.

In one embodiment, one or more phase separation behavior modifyingagents are selected to induce phase separation when the concentrationsof said components are not sufficient to induce phase separation withoutthe presence of said modifying agents.

In one embodiment, one or more phase separation behavior modifyingagents are selected to:

-   -   a) reduce fluctuations in the rate of domain coalescence; or    -   b) increase or decrease the rate of domain coalescence.

In one embodiment, one or more phase separation behavior modifyingagents are selected to:

-   -   a) reduce fluctuations in the flow rate of the first phase        solution within the porous material;    -   b) reduce fluctuations in the flow rate of the second phase        solution within the porous material;    -   c) increase the flow rate of the first phase solution within the        porous material;    -   d) decrease the flow rate of the first phase solution within the        porous material;    -   e) increase the flow rate of the second phase solution within        the porous material; or    -   f) decrease the flow rate of the second phase solution within        the porous material.

In one embodiment, one or more phase separation behavior modifyingagents are selected to:

-   -   a) reduce fluctuations in the partitioning of said target        analytes between the first phase solution and the second phase        solution;    -   b) increase the partitioning of said target analytes to the        first phase solution;    -   c) decrease the partitioning of said target analytes to the        first phase solution;    -   d) increase the partitioning of said target analytes to the        second phase solution; or    -   e) decrease the partitioning of said target analytes to the        second phase solution.

In one embodiment, the device is further integrated with a second devicesuch that a leading fluid within the porous material would transfer intosaid second device.

In one embodiment, the present invention also provides a method of usingthe device for isolating or concentrating one or more target analytesfrom a sample solution.

In one embodiment, the target analytes isolated or concentrated on theporous material are extracted to generate an extract solution to besubsequently used in another method or process.

Throughout this application, it is to be noted that the transitionalterm “comprising”, which is synonymous with “including”, “containing” or“characterized by”, is inclusive or open-ended, and does not excludeadditional, un-recited elements or method steps.

This invention will be better understood by reference to the exampleswhich follow. However, one skilled in the art will readily appreciatethat the examples provided are merely for illustrative purposes and arenot meant to limit the scope of the invention which is defined by theclaims following thereafter.

EXAMPLES Example 1—Using Dextran to Reduce Volume Ratio Fluctuations ina Device for Detecting Pathogens (S. mutans) in Patient Samples

Preparation of sample pad for LFA: Fiberglass porous paper sheets werecut into 0.5 cm×4 cm rectangles. The formulated ATPS components, 20%(w/w) PVP and 18.5% (w/w) sodium cholate were pipetted onto thefiberglass porous paper. The above porous papers with ATPS were thendried in a lyophilizer for 2 hours first. 10% (w/w) dextran was thenpipetted onto the fiberglass porous paper as the phase separationbehavior modifying agent onto the paper segments. PEG solution (in DIH₂O) was added to each porous material. 50 μl of a Tris-bufferedsolution containing 2% bovine serum albumin (BSA), and 0.1% PEG, 20 mMTris pH 7.5 respectively) was added immediately adjacent to the firstsolution.

The above porous papers with ATPS and the phase separation behaviormodifying agent were then dried in a lyophilizer for 2 hours. The ATPSwithin porous papers were then placed in an indicator-containing(colloidal gold) buffer solution in PBS (overall pH 7.4), resulting incapillary action-mediated flow. The dextran solution is applied to oneend of the porous paper so that it creates a gradient concentrationalong the diffusion direction:

The purpose of the dextran (phase separation behavior modifying agent)is to stabilize (i.e. reduce fluctuations) the volume ratio of the ATPSduring variable fluid flow rates, making the test more robust. Thegradient of dextran is important so that the volume ratio is still largeenough by the time the fluid flow reaches the end of the porousmaterial. Place paper segments under low pressure vacuum for 2 hours todehydrate.) Multiple layers of porous papers with the dehydrated ATPScomponents and phase separation behavior modifying agent are assembledtogether.

Preparation of LFA test strip: 1) anti-S. mutans antibody at aconcentration of 1 mg/mL, and 2) Protein (Bovine Serum Albumin, BSA) ata concentration of 0.2 mg/ml were added on the test strip. Colloidalgold nanoparticles were conjugated to the anti-S. mutans antibody asdirected by manufacturer instructions. This conjugate was then driedonto the conjugating pad material using a lyophilizer. The absorbent padconsisted of untreated paper.

The LFA test strip was integrated with sample pad, i.e., the porousdevice/component with the dehydrated ATPS components and the phaseseparation behavior modifying agent. The porous device/component and theLFA component are placed into an appropriate housing such that thecomponents are held in place.

Detection using LFA: The fiberglass porous paper dehydrated with ATPScomponents and the phase separation behavior modifying agent, a blankpaper (without ATPS or modifying agent) and a control paper (ATPSwithout modifying agent) were dipped in a same solution of saliva, whichwas in a PBS buffer solution at pH 7.4. After the sample solution flowedthrough the porous material and through the LFA test for 2 min, another2 mins was needed to develop the test results. The presence or absenceof a test line by visual observation was used to determine the resultsof the diagnostic. The result is summarized in Table 1 below:

TABLE 1 Intensity of test line ATPS with Control Paper Blank paper(without modifying (ATPS without ATPS or modifying agent modifyingagent) agent) Intensity of test line 10× 3× 1×

Table 1 clearly shows that adding phase separation behavior modifyingagents to ATPS can increase the intensity of test line, indicating thatthe concentration of the target analyte is increased significantly.

Example 2—Using Polysorbate and Sucrose to Increase Food Allergen(Lupine) Partition in a Detecting Device

Preparation of sample pad for LFA: Fiberglass porous paper sheets werecut into 0.5 cm×4 cm rectangles. The formulated ATPS components, 20%(w/w) PVP and 18.5% (w/w) sodium cholate were pipetted onto thefiberglass porous paper. 5% (w/w) polysorbate and 5% (w/w) sucrose werepipetted onto the porous fiberglass paper as the phase separationbehavior modifying agent. PEG solution (in DI H₂O) was added to eachporous material. 50 μl of a Tris-buffered solution containing 2% bovineserum albumin (BSA), and 0.1% PEG, 20 mM Tris pH 7.5 respectively) wasadded immediately adjacent to the first solution.

The above porous papers with dehydrated ATPS components and the phaseseparation behavior modifying agent were then dried in a lyophilizer for1 hour. The ATPS-dehydrated porous papers were then placed in anindicator-containing (colloidal gold) buffer solution in PBS (overall pH7.4), resulting in capillary action-mediated flow. The polysorbate andsucrose were applied so that it creates an even distribution of thecomponents on the porous material. Multiple layers of porous papers withthe dehydrated ATPS components and the phase separation behaviormodifying agent were assembled together.

The purpose of the polysorbate and sucrose is to increase food allergentarget partitioning into the first phase. Then place paper segments inan oven at 37 degrees Celsius for 28 hours to dehydrate.

Preparation of LFA test strip: 1) anti-lupine antibody at aconcentration of 1 mg/ml, and 2) Protein (Bovine Serum Albumin, BSA) ata concentration of 0.2 mg/ml were added on the test strip. Colloidalgold nanoparticles were conjugated to anti-lupine antibody as directedby manufacturer instructions. This conjugate was then dried onto theconjugating pad material using a lyophilizer. The absorbent padconsisted of an untreated paper.

The LFA test strip was first integrated with the sample pad, i.e., theporous material for tests food allergen (lupine) and then integratedwith a food matrix grinder/processor. The integrated device should beassembled such that the flow of the sample solution will go from thefood matrix grinder/processor down into the porous component, then flowforward further through the porous material where it will resolubilizethe ATPS components and the phase separation behavior modifying agentswhich will induce phase separation and facilitate food allergenpartition into the leading first phase, and finally into the LFAcomponent. All of the components were integrated into the appropriatecassette/housing to hold the components in place.

Detection of food allergen using LFA. The food samples of interest wereplaced into the food matrix grinder/processor portion of the device. Thefood matrix grinder/processor was manually operated to break down thefood matrix. The fiberglass porous paper dehydrated with ATPS componentsand the phase separation behavior modifying agent, a blank paper(without ATPS or modifying agent) and a control paper (ATPS withoutmodifying agent) were dipped in food matrix sample in a PBS buffersolution at pH 7.4. After the sample solution flowed through theremaining device for 2 min, another 2 min was needed to develop the testresults. The presence or absence of a test line by visual observationwas used to determine the results of the diagnostic. The result issummarized in Table 2 below:

TABLE 2 Intensity of test line ATPS with Control Paper Blank paper(without modifying (ATPS without ATPS or modifying agent modifyingagent) agent) Intensity of test line 8× 2× 1×

Table 2 clearly shows that adding phase separation behavior modifyingagents to ATPS can increase the intensity of test line, indicating thatthe concentration of the target analyte is increased significantly.

Example 3—Using Potassium Phosphate to Increase Total Phase SeparationVolume in a Device for Sample Solution Purification and DNAAmplification

Porous hydrogel material was cut into cylindrical shape with dimensionsof 2 cm radius and 6 cm height. The porous hydrogel component wasintegrated into an appropriate cassette/housing. The device can exposethe bottom of the hydrogel as well as the top of the hydrogel.

The ATPS components, 20% (w/w) PVP and 18.5% (w/w) sodium cholate, 10%(w/w) potassium phosphate as the phase separation behavior modifyingagent were mixed with a blood sample solution containing the DNAsegments of interest in a PBS buffer solution at pH 7.4. The purpose ofthe potassium phosphate is to induce macroscopic phase separation at anearlier point (more upstream) during the flow of solutions through theporous material, resulting in a great volume undergoing phase separationand a higher volume of solution that is able to be collected at the endof the process. The final volume should be about 120% of the volume thatthe hydrogel can hold. The sample solution was placed in an opencontainer or tube that can hold the tapered porous device in an uprightposition. A blank hydrogel material (without ATPS components ormodifying agent) and a control hydrogel material (ATPS without modifyingagent) were used for comparison.

The bottom and the top of the device were opened to expose the bottomand top of the tapered porous hydrogel. The device was placed into themixture of sample solution and ATPS components. The liquid of themixture is in contact with the bottom of the porous hydrogel.

Extraction of sample solution: After the solution flowed to the top ofthe porous hydrogel for 2 min, the purified sample solution wasextracted by pipetting it out of the top region of the porous hydrogel.The extracted sample solution is then ready for DNA amplification. Theyield of purified sample was analyzed by UV-Vis spectrophotometry. Theresult is summarized in Table 3 below.

TABLE 3 Fold of concentration of DNA using the present invention ATPSwith Control hydrogel Blank hydrogel modifying (ATPS without (withoutATPS or Test agent modifying agent) modifying agent) Yield of DNA (%)98% 50% 7% Fold of 100-fold 50-fold 1-fold Concentration of DNA

What is claimed is:
 1. A method for isolating or concentrating one ormore target analytes from a sample solution, comprising the steps of:(a) embedding a porous material with one or more phase separationbehavior modifying agents and aqueous two-phase system (ATPS)components, wherein said components form a first phase solution and asecond phase solution; (b) obtaining a sample solution containing thetarget analytes; and (c) contacting the sample solution with the porousmaterial embedded with the one or more phase separation behaviormodifying agents and the aqueous two-phase system (ATPS) components,wherein, when the sample solution flows through the porous material, anaqueous two-phase system comprising said first phase solution and saidsecond phase solution is generated, wherein the separation behavior ofthe two phase solutions is altered by said phase separation behaviormodifying agents; and wherein the target analytes are isolated orconcentrated on said porous material.
 2. The method of claim 1, whereinthe step (a) comprising the steps of: (i) pipetting the aqueous twophase system components onto the porous material; (ii) drying the porousmaterial with the aqueous two phase system components in a lyophilizer;(iii) pipetting the phase separation behavior modifying agent onto theporous material, wherein the phase separation behavior modifying agentis applied to one end of the porous material such that a gradientconcentration is created; and (iv) drying the porous material with theATPS components and the phase separation behavior modifying agent. 3.The method of claim 2, wherein the phase separation behavior modifyingagent is 10% (w/w) dextran, and the aqueous two-phase system (ATPS)components comprise 20% (w/w) PVP and 18.5% (w/w) sodium cholate.
 4. Themethod of claim 1, further comprising the steps of: (d) extracting thetarget analytes isolated or concentrated on said porous material togenerate an extract solution; (e) subjecting the extract solution todetection, analytical or diagnostic procedures involving the detectionor quantification of the target analytes.
 5. The method of claim 1,wherein said one or more phase separation behavior modifying agents areselected from the group consisting of: (i) compounds carrying one ormore acidic functional groups; (ii) compounds carrying one or more aminefunctional groups; (iii) compounds carrying both hydrophilic andhydrophobic groups; (iv) chaotropic agents; and (v) kosmotropic agents.6. The method of claim 5, wherein said compounds carrying one or moreacidic functional groups are selected from the group consisting ofsaccharides, polysaccharides, sorbates, polysorbates, carboxylates,polycarboxylates, phosphates, polyphosphates, sulfates, polysulfates andpolyols.
 7. The method of claim 5, wherein said compounds carrying oneor more amine functional groups are selected from the group consistingof amines, polyamines, amine salts and polyamine salts.
 8. The method ofclaim 5, wherein said compounds carrying both hydrophilic andhydrophobic groups are selected from the group consisting of lipids,surfactants, nucleic acids, hormones, proteins, and amino acids.
 9. Themethod of claim 1, wherein said phase separation behavior modifyingagents comprise sucrose, potassium phosphates, dextran, Tween-20 orTriton-X114.
 10. A method for isolating or concentrating one or moretarget analytes from a sample solution, comprising the steps of: (a)providing one or more phase separation behavior modifying agents andaqueous two-phase system (ATPS) components, wherein said components forma first phase solution and a second phase solution; (b) embedding aporous material with at least a portion of one or more of the phaseseparation behavior modifying agents and the aqueous two-phase systemcomponents; (c) combining a remaining portion of one or more of thephase separation behavior modifying agents and the aqueous two-phasesystem components with a sample solution containing the target analytesto form a mixed solution; and (d) adding the mixed solution to theporous material, wherein, when the mixed solution flows through theporous material, an aqueous two-phase system comprising said first phasesolution and said second phase solution is generated, wherein theseparation behavior of the two phase solutions is altered by said phaseseparation behavior modifying agents; and wherein the target analytesare isolated or concentrated on said porous material.
 11. The method ofclaim 10, wherein the step (b) comprises the steps of: (i) embedding aportion of the phase separation behavior modifying agents in the porousmaterial containing the first phase solution and the second phasesolution of the aqueous two-phase system components; and (ii) dehydratethe porous material; and wherein the step (c) comprises the step of:(iii) combining a remaining portion of the phase separation behaviormodifying agents with a sample solution containing the target analytesto form the mixed solution.
 12. The method of claim 10, wherein the step(b) comprises the steps of: (i) embedding a portion of the phaseseparation behavior modifying agents in the porous material containingcomponents of the first phase solution of the aqueous two-phase systemcomponents; and (ii) dehydrate the porous material; and wherein the step(c) comprises the step of: (iii) combining a remaining portion of thephase separation behavior modifying agents with components of the secondphase solution of the aqueous two-phase system components to form themixed solution.
 13. The method of claim 10, wherein the step (b)comprises the steps of: (i) embedding a portion of the phase separationbehavior modifying agents in the porous material containing componentsof the second phase solution of the aqueous two-phase system components;and (ii) dehydrate the porous material; and wherein the step (c)comprises the step of: (iii) combining a remaining portion of the phaseseparation behavior modifying agents with components of the first phasesolution of the aqueous two-phase system components to form the mixedsolution.
 14. The method of claim 10, wherein the step (b) comprises thesteps of: (i) embedding a portion of the phase separation behaviormodifying agents in the porous material; and (ii) dehydrate the porousmaterial; and wherein the step (c) comprises the step of: (iii)combining a remaining portion of the phase separation behavior modifyingagents with components of the first phase solution and components of thesecond phase solution of the aqueous two-phase system components to formthe mixed solution.
 15. The method of claim 10, further comprising thesteps of: (f) extracting the target analytes isolated or concentrated onsaid porous material to generate an extract solution; (g) subjecting theextract solution to detection, analytical or diagnostic proceduresinvolving the detection or quantification of the target analytes. 16.The method of claim 10, wherein said one or more phase separationbehavior modifying agents are present in some regions of the porousmaterial, and not present in other regions of the porous material. 17.The method of claim 10, wherein said one or more phase separationbehavior modifying agents are evenly distributed on said porous materialor present in an increasing amount from one end to the other end of saidporous material.
 18. The method of claim 10, wherein said one or morephase separation behavior modifying agents are present in varyingamounts in different regions of the porous material.
 19. The method ofclaim 10, wherein said one or more phase separation behavior modifyingagents are selected from the group consisting of: (i) compounds carryingone or more acidic functional groups; (ii) compounds carrying one ormore amine functional groups; (iii) compounds carrying both hydrophilicand hydrophobic groups; (iv) chaotropic agents; and (v) kosmotropicagents.
 20. The method of claim 19, wherein said compounds carrying oneor more acidic functional groups are selected from the group consistingof saccharides, polysaccharides, sorbates, polysorbates, carboxylates,polycarboxylates, phosphates, polyphosphates, sulfates, polysulfates andpolyols.
 21. The method of claim 19, wherein said compounds carrying oneor more amine functional groups are selected from the group consistingof amines, polyamines, amine salts and polyamine salts.
 22. The methodof claim 19, wherein said compounds carrying both hydrophilic andhydrophobic groups are selected from the group consisting of lipids,surfactants, nucleic acids, hormones, proteins, and amino acids.
 23. Themethod of claim 10, wherein said phase separation behavior modifyingagents comprise sucrose, potassium phosphates, dextran, Tween-20 orTriton-X114.